Isolation and Symbiotic Characteristics of Two Tn5-Derived Phage-Resistant <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> Strains that Nodulate Soybean

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.     

The use of transposons for the mutagenesis of R. phaseoli and R. japonicum

Plasmid pSUp2011 has been used to transfer transposon Tn5 into the cells of R. japonicum 110 and R. phaseoli 693. Transposition of Tn5 into the chromosomes of R. japonicum and R. phaseoli has been demonstrated,resulting in isolation of auxotrophic and symbiotic mutants of both strains. The frequencies of selected auxotrophic mutations have reached 4% in R. japonicum 110 and 0.6% in R. phaseoli 693. Streptomycin resistance gene locating on Tn5 has been found to be phenotypically expressed in R. japonicum 110 and R. phaseoli 693 cells.     

Prevalence and distribution of pig helminths in the Dongting Lake Region (Hunan Province) of the People's Republic of China

The prevalence of helminths in pigs was investigated in five rural communities situated on the embankment of Dongting Lake in Zhiyang County, Hunan Province, People's Republic of China, in an area known to be endemic for Schistosoma japonicum. The helminth prevalences identified on the basis of faecal egg count analysis were: Oesophagostomum spp. (86.7%), Ascaris suum (36.7%), Metastrongylus spp. (25.8%), Strongyloides spp. (25.8%), Trichuris suis (15.8%), Globocephalus spp. (6.7%), Gnathostoma spp. (4.2%), Schistosoma japonicum (5.0%) and Fasciola spp. (1.3%). Post mortem examinations of a small number of pigs depositing eggs of different helminth species revealed the presence of Oesophagostomum dentatum, O. quadrispinulatum, A. suum, Metastrongylus apri, M. pudendotectus, T. suis, G. hispidum and Ascarops dentata. Prevalences of all helminths, with the exception of Oesophagostomum spp., were higher in young pigs (< 8 months old) compared with adult pigs. Prevalences of trematodes were very low, especially for S. japonicum which had decreased dramatically compared with previous reports from this area of P.R. China, whereas prevalences of nematodes were generally in agreement with those reported from other Yangtze River Provinces. Results from helminth prevalence studies in pigs, conducted in other provinces of P.R. China between 1987 and 1997, are presented and discussed. It was concluded that a government helminth control programme, implemented in 1995 to control S. japonicum infection in pigs in Hunan Province, may have resulted in a greatly reduced prevalence of S. japonicum in pigs in this region.     

Expression and functional roles of Bradyrhizobium japonicum genes involved in the utilization of inorganic and organic sulfur compounds in free-living and symbiotic conditions

Strains of Bradyrhizobium spp. form nitrogen-fixing symbioses with many legumes, including soybean. Although inorganic sulfur is preferred by bacteria in laboratory conditions, sulfur in agricultural soil is mainly present as sulfonates and sulfur esters. Here, we show that Bradyrhizobium japonicum and B. elkanii strains were able to utilize sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources for growth. Expression and functional analysis revealed that two sets of gene clusters (bll6449 to bll6455 or bll7007 to bll7011) are important for utilization of sulfonates sulfur source. The bll6451 or bll7010 genes are also expressed in the symbiotic nodules. However, B. japonicum mutants defective in either of the sulfonate utilization operons were not affected for symbiosis with soybean, indicating the functional redundancy or availability of other sulfur sources in planta. In accordance, B. japonicum bacteroids possessed significant sulfatase activity. These results indicate that strains of Bradyrhizobium spp. likely use organosulfur compounds for growth and survival in soils, as well as for legume nodulation and nitrogen fixation.     

Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms

Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.     

Phylogenetic diversity and symbiotic functioning in mungbean (Vigna radiata L. Wilczek) bradyrhizobia from contrast agro-ecological regions of Nepal

Nepal consists wide range of climatic and topographical variations. Here, we explored the phylogeny of native mungbean bradyrhizobia isolated from different agro-ecological regions of Nepal and accessed their nodulation and nitrogen fixation characteristics. Soil samples were collected from three agro-ecological regions with contrasting climate and topography. A local mungbean cultivar, Kalyan, was used as a trap plant. We characterized isolates based on the full nucleotide sequence of the 16S rRNA, ITS region, and nodA genes; and partial sequences of nodD1 and nifD genes. We found 50% of isolates phylogenetically related to B. yuanmingense, 13% to B. japonicum, 8% to B. elkanii, and 29% to novel phylogenetic origin. Results of the inoculation test suggested that expression of different symbiotic genes in isolates resulted in different degrees of symbiotic functioning. Our results indicate B. yuanmingense and novel strains are more efficient symbiotic partners than B. elkanii for the local mungbean cv. Kalyan. We also found most mungbean rhizobial genotypes were conserved across agro-ecological regions. All the strains from tropical Terai region belonged to B. yuanmingense or a novel lineage of B. yuanmingense, and dominance of B. japonicum related strains was observed in the Hill region. Higher genetic diversity of Bradyrhizobium strains was observed in temperate and sub-tropical region than in the tropical region.     

Diversity among Bradyrhizobium isolates nodulating yardlong bean and sunnhemp in Guam

AIMS: To isolate and characterize bradyrhizobia that nodulate yardlong bean and sunnhemp in Guam. METHODS AND RESULTS: Bradyrhizobia populations that nodulate yardlong bean and sunnhemp in Guam were examined for genetic diversity and their relatedness to Bradyrhizobium japonicum and B. elkanii reference strains. Genomic DNA of 58 isolates of Bradyrhizobium spp. was hybridized with B. japonicum nodY and B. elkanii nodK genes. Based on the hybridization patterns, the isolates were classified into three nodY-nodK hybridizing groups. Group I comprised the majority of the isolates and hybridized with nodY whereas group II isolates hybridized with nodK. The group III isolates, that did not hybridize with either nodY or nodK, formed nitrogen-fixing nodules on cowpea but did not nodulate soybean. DNA sequence analysis of a 280-bp fragment of the variable region of the 16S rRNA gene of a few group III isolates showed that these isolates were more similar to Bradyrhizobium spp. than to B. japonicum or B. elkanii. CONCLUSIONS: The majority of the isolates nodulating yardlong bean and sunnhemp in Guam are similar to B. japonicum, although some isolates are similar to Bradyrhizobium spp. that nodulate a miscellaneous group of legumes including cowpea. SIGNIFICANCE AND IMPACT OF THE STUDY: Since both yardlong bean and sunnhemp are nodulated by a range of bradyrhizobia, selection of superior strains may be based on nodulation effectiveness on both legumes.     

Bacteriocin-like substances produced by Rhizobium japonicum and other slow-growing rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80 degrees C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.     

Variation in the nod gene RFLPs, nucleotide sequences of 16S rRNA genes, Nod factors, and nodulation abilities of Bradyrhizobium strains isolated from Thai Vigna plants

The analysis of nod genes and 16S rRNA gene regions, Nod factors, and nodulation abilities of Brady rhizobium strains isolated from tropical Thai Vigna species is reported. A total of 55 Bradyrhizobium strains isolated from two cultivated and six wild Vigna species growing in central and northern Thailand were evaluated. Thai Vigna spp. Bradyrhizobium strains showed higher levels of nod gene RFLP diversity compared with Thai soybean Brady rhizobium strains or temperate strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Analysis of the 16S rRNA gene region using selected strains also suggests a high genetic diversity of the Thai Vigna-Bradyrhizobium association. Based on thin-layer chromatography analysis, Nod factors produced by tropical Thai Vigna spp. Brady rhizobium strains are more diverse than temperate Japanese and US strains of B. japonicum and B. elkanii. Thai Vigna spp. Bradyrhizobium strains showed variation in nodulation ability and affinity, estimated by the number of normal nodules versus green nodules in an inoculation study. There are some Bradyrhizobium-host combinations that could not form any nodules, suggesting that some genetic differentiation has evolved in their host range. However, most of the Thai Vigna spp. Bradyrhizobium strains formed nodules on the cultigens soybean (Glycine max), mungbean (Vigna radiata), azuki bean (Vigna angularis), and cowpea (Vigna unguiculata). This is the first study on Bradyrhizobium strains associated with a range of cultivated and wild Vigna and reveals that these Bradyrhizobium strains are diverse and may provide novel sources of useful variation for the improvement of symbiotic systems.     

A mutant of Escherichia coli capable of utilizing 3-deoxy-d-manno-2-octulosonic acid as sole carbon source

Abstract The hypothesis that rifampicin resistance mutations (possibly leading to altered RNA polymerases) have a pleiotropic effect on symbiotic nitrogen fixation was tested using the Rhizobium japonicum -soybean symbiosis. A total of 20 spontaneous rifampicin-resistant mutants of R. japonicum strain 110 were analyzed biochemically. RNA polymerase assays revealed that the enzyme from 15 mutants was indeed rifampicin-insensitive. Two of these mutants were found to possess an enzyme with an electrophoretically altered β subunit. All rifampicin-resistant mutants were able to form nodules on soybeans and fix nitrogen symbiotically; free-living nitrogen fixation under microaerophilic culture conditions was also unaffected.     

High-frequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum.

More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques. Wild-type R. japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants. The symbiotic traits of 133 isolates from nine treatment groups were determined. Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif-. The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe. None of these mutants showed any detectable loss of plasmid DNA.     

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.     

Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain

As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.     

The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins

The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Bradyrhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N-terminal domain is not essential for NifA function in R.I. bv. trifolii.     

Photosynthetic Bradyrhizobium Sp. strain ORS285 synthesizes 2-O-methylfucosylated lipochitooligosaccharides for nod gene? Dependent interaction with Aeschynomene plants

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C(18:1)-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N(2)-fixing nodules on both the roots and stems of Aeschynomene afraspera.     

Bradyrhizobium japonicum glnB, a putative nitrogen-regulatory gene, is regulated by NtrC at tandem promoters.

The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from Klebsiella pneumoniae and Escherichia coli. The gene is located directly upstream from glnA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonicum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.     

Identification of the host-range DNA which allows Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas

Summary The special ability of Rhizobium leguminosarum strain TOM to nodulate cv. Afghanistan peas had previously been shown to be determined by the symbiotic plasmid, pRL5JI, of this strain. A region of pRL5JI, 2.0 kb in size, was found to confer the ability to nodulate cv. Afghanistan peas when transferred to strains of R. leguminosarum which normally fail to nodulate this host. This region of pRL5JI, responsible for the extension of host-range, was closely linked to, but did not include, the genes required for root hair curling. Although extensive homology has been found between the R. leguminosarum nod genes on pRL5JI and those on the normal symbiotic plasmid pRL1JI, a fragment from the 2.0 kb region involved in nodulation of cv. Afghanistan has been identified, which was not homologous to DNA in strains which do not nodulate cv. Afghanistan.     

Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah soil

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N(2)-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.     

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.